Past, present and future of the two-spotted stink bug (Perillus bioculatus) in Europe revealed by citizen science

The introduction of the Nearctic predaceous stink bug species, (Perillus bioculatus) was attempted multiple times in various countries throughout Europe to mitigate the damage caused by the invasive and harmful pest species, the Colorado potato beetle (Leptinotarsa decemlineata). Though these attempts were thought to be unsuccessful for decades, more recent data elucidated that the species have established small self-sustaining populations in the Balkans Peninsula, Southern Russia, and Türkiye and recently began to expand. In the past years, the European range of the species reached Eastern Europe. After the first individuals were found in Hungary in October 2023 a citizen science campaign was launched to investigate the distribution of the species in the country. By June 2024 it became evident that the species is established throughout the country. Furthermore, observations regarding beetle larvae and moth caterpillars as alternative prey were reported supporting the previous assumptions that the naturalization and expansion of the species in Europe is facilitated by dietary drift. Here, we summarize the knowledge on the European presence of the two-spotted stink bug and formulate hypotheses regarding its future distribution and the impact of the species on the insect communities of the newly colonized areas.

Russian Federation 15 , Serbia 22,23 and Türkiye 18,24 .The survival, naturalization and range expansion of the species are hypothesized to be associated with both climate change and the dietary drift observed 15,16,21,23 .Latter means the utilization of alternative prey species as recorded first in the case of the ragweed leaf beetle, Zygogramma suturalis (Fabricius, 1775) and the olive-shaded bird-dropping moth, Tarachidia candefacta (Hübner, 1813), both species are feeding on common ragweed (Ambrosia artemisifolia) and were introduced to the southern parts of Russia to control the populations of this invasive weed species 15 .Later predation of the species was observed on leaf beetle species Chrysomela populi Linnaeus, 1758 and Chrysolina herbacea (Dufschmid, 1825) 24,25 , both species are native to Europe.Most recently the two-spotted stink bug was reported to be feeding on the larvae of the ragweed specialist leaf beetle species, Ophraella communa LeSage, 1986 23 .As the two-spotted stink is of very characteristic and conspicuous appearance, i.e., can be identified securely on sight, the increasing activity of citizen scientists resulted in numerous records for the species recently submitted on platforms like iNaturalist.org 26 and on the Facebook social media platform.The latter revealed the presence of the species in Hungary in October 2023.As the invaluable contributions of citizen scientists were proven by the cases of multiple invasive species of characteristic appearance [e.g., 27-29], we launched a campaign in March 2024 to investigate the status of the species in Hungary.We combined the resulting data with records submitted to citizen science platforms and the available literature and analysed with the implication of geographic information systems and ecological niche modelling.Here, we present the results of this study that offered elaborate knowledge to assess the current status and formulate hypotheses regarding the future of the two-spotted stink bug in Europe.

Citizen science data
Out of the 30 submitted observations (details can be found in Supplementary Data S1) 27 included photographs of the observed insects.14 included the observation of a single individual, 15 of them reported the simultaneous presence of multiple individuals and one observation reported a mass occurrence of more than 20 individuals (Fig. 2).7 of the observations of numerous individuals did not specify the observed number but phrases 'more' or 'several' were included in the report, thus we decided to include them in this category.Most observations were made in gardens with potatoes planted (17 observations), one individual was observed inside a house in January and two observations of multiple individuals was reported from a commercial potato plantations.The single occurrence that was reported from a residential building that is accidental rather than the phenomenon of hibernation in buildings.In terms of observation of specific behaviour in 14 records no indication of such was included, i.e., the observed insect was crawling on the substrate or was stationary.In 11 cases predation was observed (Fig. 1b-c) including an observation of feeding on the caterpillar of the turnip moth, Agrotis segetum (Dennis & Schiffmüller, 1775) (Lepidoptera: Noctuidae).One observation reported mating; in four cases, both mating and feeding were reported (Fig. 1b).
The mass occurrence was reported from the garden, and both observations in the potato plantation reported the presence of multiple individuals.Observations of specific behaviours were predominantly reported in gardens (15 observations) except a record of predation observed in potato plantations.The report on mass occurrence included the observation of predation.Instances with both mating and predation observed were reported from gardens (Fig. 2).

Distribution history of P. bioculatus in Europe
The 145 individual records compiled (Supplementary Table S2) consisted of one specimen deposited in the Hemiptera collection of HNHM, 27 previously published records from literature, and 117 citizen science records (80,7%) (Fig. 3a).From the latter 87 were acquired from citizen science platforms and 30 resulted by the campaign launched by the authors.The five occurrences recorded before 2010 originated from the museum specimen and literature data.Seventeen occurrences were recorded between 2010 and 2020 of which 11 were acquired from the citizen science database iNaturalist.org(64.7%) and 6 were published in articles regarding the European presence of the species.After 2020, 17 records were found in the literature and 106 occurrences were provided by citizen science contributors (86.2% of the 123 post-2020 records).
The compiled distribution data shows that P. biolculatus is established in Bulgaria, Greece, Hungary, Moldavia, Russia, Serbia, Türkiye, and Ukraine.From Hungary, the presence of the species was not indicated until The pinned specimen found in the collection of HNHM represents the earliest known specimen of the species collected in in Europe -presumably in a natural or semi-natural habitat -after the repeated and rather unsuccessful attempts of introduction (Fig. 3a).The pre-2010 records originate from Bulgaria, Greece and Serbia corroborating the hypothesis that species have survived in small populations throughout the Balkans Peninsula and Türkiye (Fig. 3b).Between 2010 and 2020 the species were recorded additionally in Moldavia, Southwest Russia and in Ukraine.The post-2020 records suggest both the local increase of abundance in the previously colonized regions and an expansion in the north-northwestern direction (Fig. 3b).When interpreting the data using the Koeppen-Geiger climatic classification dataset, the records are originating predominantly from arid www.nature.com/scientificreports/cold steppes (BSk), temperate (Cfa) and cold (Dfa) zones with no dry season and hot summer (Supplementary Fig. S3) that corresponds the conditions found in the native area of the species.

Results of the ecological niche modelling
The area under the curve in the threshold-independent receiver operation characteristic (AUC) evaluation showed a very good performance of the constructed models with an average value of 0.916 (standard deviation: 0.004).The variables with highest relative contribution to the model were the maximum temperature of the warmest month (bio5: 41.0%), the minimum temperature of the coldest month (bio6: 19.4%), and the precipitation of the driest month (bio14: 17.5%) and the Jackknife analysis of predictor importance revealed that the variable 'maximum temperature of the warmest month' (bio5) provides the most useful information when used in isolation and the precipitation of the driest month (bio14) when omitted.The variables 'annual mean temperature' (bio1) and 'minimum temperature of the coldest month' (bio6) were found to be the second and third most important predictors in both cases (Supplementary Fig. S4).The response curves revealed that there is a positive correlation between the annual mean temperature (bio1) and the occurrence of P. bioculatus, contrastingly, a negative response is shown for low and high values of the temperatures of the warmest and coldest months (bio5, bio6) (Supplementary Fig. S4).The models showed that from the regions of Europe currently not inhabited by P. bioculatus the most suitable (probability of habitat suitability ≥ 0.5) are the foothill areas of the Carpathians and the Eastern Alps (Burgenland and Lower Austria federal states of Austria; South Bohemian region of the Czech Republic; lower parts of Bratislava, Trnava, Trenčín, Nitra, Banská Bystrica and Košice regions of Slovakia; Zakarpatska region of Ukraine) the northern, pre-alpine part of Italy (mostly Friuli-Venezia Giulia, Veneto, Trentino-Alto Adige, Emili-Romagna, Lombardy, and Piedmont regions), southern and middle parts of France (parts of Nouvelle-Aquitaine and Occitanie regions) and the northeastern parts of the Iberian Peninsula (parts of Catalonia, Aragon, and Castille and Leon regions) (Fig. 4).Moderately and less suitable areas (probability of habitat suitability < 0.5) are found in Central Italy, north of France, Southern Germany, and Poland.The projection of the Koeppen-Geiger climatic classification maps implies a cut-off value of 0.5 for suitability to explore the most suitable predicted area corresponding to the projections done for the existing records in terms of climatic zones with the addition of temperate regions with no dry season and warm summer (Cfb) (Supplementary Fig. S3).

Discussion
A citizen science campaign was launched after the first reported occurrences of the Nearctic two-spotted stink bug, Perillus bioculatus in Hungary (October 2023).More than half of the 30 submitted records indicated the presence of multiple individuals at the site of observation and on one occasion mass occurrence (> 20 individuals) was reported (Supplementary Data S1), thus it is to be concluded that species is established and widely distributed in Hungary.Combining the knowledge on the distribution of the species derived from museum specimens, literature data, citizen science platforms and data of the citizen science campaign revealed that P. bioculatus is widely established in the southeastern parts of Europe and is suspected to be expanding in the north-northwestern direction (Fig. 3b).The environmental niche models built from these data suggest that the Fig. 4. Habitat suitability in Europe resulted from the average of 10 equally robust ENM models (values ranging between 0-1 where 0 is completely unsuitable and 1 is 100% suitable) (map was generated using Maxent software, version 3.4.3).
distribution of P. bioculatus is closely associated with three bioclimatic variables, the temperature of the warmest, minimum temperature of the coldest months, and the precipitation of the driest month.Response curves revealed that there is a positive correlation between higher mean temperatures and the suitability of the habitats, however, high values of maximum temperatures in the warmest (bio5) and minimum temperatures of the coldest months (bio6) may negatively affect it (Supplementary Fig. S4).The former can be explained by the positive correlation between the mean temperature and the effective temperature needed to complete the life cycle of the insect.The negative response to higher temperatures in the coldest months is suspected to be associated with the facultative diapause of the insect in the local climate which is crucial in terms of the survival of the overwintering adults due to the lack of suitable prey in the cold periods 16 .This suggestion supports the conclusions of studies that attributed the unsuccessful naturalization of P. bioculatus to the asynchronous seasonal cycle of predator and prey in Europe 15 .On the other hand, high temperatures attributed to climate change in the warmest and coldest months may induce phenological shifts that impede the completion of the nymphal development and successful formation of diapause before the disadvantageous periods of the year [30][31][32] .The results of the ENM correspond to the conditions characteristic to the climatic zones where the species is currently distributed, thus the species will most likely invade other parts of the continent with bioclimatic environments similar to its current distribution.Most of the observations were reported from an environment with potatoes planted where the primary prey of P. bioculatus viz. the Colorado potato beetle (Leptinotarsa decemlineata) is to be found (Fig. 2).In fifteen instances predation was observed including a new prey record of the caterpillar of the noctuid moth, Agrotis segetum (Fig. 5a), a widely distributed lepidopteran species that is considered as agricultural pest.Furthermore, an external record was shared with the senior author of a captive adult individual feeding on the larvae of the chrysomelid beetle, Galeruca tanaceti (Fig. 5b).Though P. bioculatus was known as an oligophagous predator for decades, novel data suggest that the successful naturalization of the species in Europe is associated with a dietary drift, i.e., the utilization of alternative preys at least in adult stage (Supplementary Table S5).Laboratory experiments concluded that feeding the nymphs with alternative prey may cause higher malformation and death rates in the course of development 25 , thus L. decemlineata is the most suitable prey for the nymphal stages of P. bioculatus.However, according to the prey records it is suggested that adults effectively utilise alternative food sources, i.e., larvae of other chrysomelid beetle and caterpillars of noctuid moth species, in the absence of the www.nature.com/scientificreports/Colorado potato beetle 5,24,25 .The rapid expansion and dietary drift raise the question of how the establishment and expansion of P. bioculatus will impact European insect faunas with an emphasis on the competitive pressure put on native asopine true bugs and the prospective prey species of nature conservation importance.The topicality of the question can be explained by the case of Harmonia axyridis.This Asian ladybeetle species was introduced to Europe as an effective biocontrol agent of aphids, but after its naturalisation and expansion became a strong competitor and intraguild predator of native coccinellids causing serious ecological problems 33,34 .The first aspect was investigated by reviewing the habitat utilization and prey preference data available for European Asopiane (Supplementary Table S6).The compiled literature data suggests that P. bioculatus can be the competitor of most if not all native predatory stink bugs because the European fauna predominantly consists of generalist species both in terms of habitat and prey preferences.However, it must be noted that the prerequisite of this scenario is the infiltration of natural and semi-natural habitats where these species are predominantly found.
The citizen science data suggests that the species is currently found in anthropogenic habitats, i.e. gardens and plantations.This can be explained by the presence of the primary prey species in these habitats and in part by the more intensive presence of contributors in such environments.However, the increasing population densities and literature data (e.g.,ref 15,22 ) suggest that two-spotted stink will likely invade natural and semi-natural habitats too.
The second aspect resulted in the invasion of non-anthropogenic habitats inhabited by various species allied to known prey, i.e., chrysomelid beetles and noctuid moths.These groups are of nature conservation importance at least regionally because of the limited distribution or host plant specificity of a relatively high proportion of the included species.Notable examples are the case of Serbian and Romanian noctuid moths where 35.67 and 53,84% of the species meet at least the "Near Threatened" criteria of the IUCN Red List 35 .Furthermore, a study on the Italian Chrysomelidae fauna revealed that 14.82% of the species are endemic to the Apennine Peninsula 36 .
In these countries P. bioculatus is either established or suspected to settle in the future, thus their insect faunas are potentially affected by the ongoing expansion.However, it must be stressed that this thread is merely speculative serving as a call for attention and future monitoring data may prove or refute these suggestions.
Our results reveal the established and widely distributed status of the predatory stink bug, P. bioculatus in Hungary and Eastern Europe and formulate hypotheses on an ongoing westward range expansion that presumably affects regions with moderately warm climates (Fig. 4).It must be noted that the species is apparently in the expansion phase of the invasion based on the available data and, thus not to be considered in equilibrium with the environment of the invaded area 37 .Models constructed for a species in a non-equilibrium state are less accurate than those in equilibrium because of the possibility of niche shifts during the expansion 38 .In the case of the two-spotted stink bug, it is assumed that the species has found a "home away from home" in Europe, i.e., both its realized and invaded niches are part of the fundamental niche as it was found in the cases of invasive species introduced from North America to Europe and vice versa 39 .This assumption is supported by the results of previous field experiments on rearing 4,5,15,16 along with similarities in the climate of the native and invaded ranges and the results of our ENM models [Supplementary data S3, S4].However, the possibility of further shifts in the invaded range cannot be excluded, thus we want to stress that our study is an initial assessment of the recent European invasion of the two-spotted stink bug and serves as an early warning that can be clarified or even revised based on further distribution data originating from other parts of Europe.We also corroborate the hypotheses that the successful naturalization and expansion of the species on the continent is promoted by climate change and dietary drift simultaneously as suggested by previous studies from other European countries 15,21,23 .The results of the campaign launched after the first records of the two-spotted stink bug in Hungary and the data acquired from community data-sharing platforms support the account that citizen science is a powerful tool that provides invaluable data regarding the expansions and life history characteristics of invasive species [27][28][29]34 , especially in early stages of invasion.

Citizen science data acquisition, curation and interpretation
After the first records of P. bioculatus in Hungary (5.10. 2023, Cserkút; 7. 10. 2023, Biharugra), we launched a citizen science campaign on the Facebook social media platform on 31.03.2024.The contributors were asked to report sightings of the species including photo documentation and description as detailed as possible of the circumstances of record viz.observations regarding habitat and behaviour.Until the last update (20.06.2024) 30 valid observations were submitted, 29 from various locations in Hungary and one from Senta, Serbia (Supplementary Data S1).Due to the characteristic appearance of P. bioculatus, no misidentified observations resulted, thus the three records with no photographic proof were included in the analysis.Submitted data was recorded using Microsoft Excel (.xlsx) assigning a unique identifier (PERBIO_HUN_001, PERBIO_HUN_002, etc.) to individual observations.Fields of the dataset consist of species name, individuals observed, date of observation, country, location, coordinates of location, habitat, observed behaviour (predation or copulation) and reference to submitted media (photos and videos).
To interpret resulted data the following categories were generated based on the content of the reports: • Abundance: single (1 observed individual), multiple (2-10 observed individuals), mass occurrences (> 10 observed individuals) • Habitat: garden (with potato planted), potato plantation, indoor • Behaviour: n.a.(no specific behaviour was observed, e.g., the insect was crawling on the substrate or was stationary), mating, predation, both [multiple individuals were observed either mating, feeding or both simultaneously (e.g., Fig. 2b)].
Results were visualized with Scimago Graphica software.
Vol:.( 1234567890 European distribution database of P. bioculatus -a sum of 145 records -was obtained from published literature, records on citizen science platforms (iNaturalist, izeltlabuak.hu),and records submitted via e-mail from citizen science contributors to the call published on Facebook.A summarized list of records (including data sources) can be found in Supplementary Table S2.Localities of previously attempted introductions were not included because the establishment cannot be proven.26 records from literature and citizen science observations where locality is unequivocally identifiable at least to the municipality level were georeferenced postfactum using Google Earth software.Records were grouped into three time periods (pre-2010, 2010-2020, and post-2020) to visualize and interpret range expansion.
For ecological niche modelling a combined dataset of 798 geographically unique records( 26,40 , Supplementary Table S2) including both the native and invaded range of the species following the conclusions of Broennimann & Guisan 41 .145 records originated from the invaded range (Europe) as detailed above and 653 records represented the native area (North America) of the species.Eight environmental variables, annual mean temperature (bio1), mean diurnal range (bio2), the maximum temperature of the warmest month (bio5), minimum temperature of the coldest month (bio6), annual precipitation (bio12), precipitation of the wettest (bio13) and driest (bio14) months, and elevation above sea level (bio_elev) were obtained from the WorldClim 2.0 raster dataset of 30' (~ 1 km) spatial resolution 42 to predict distribution of P. bioculatus.The variables were chosen based on the results of previous studies which concluded that these environmental dimensions are the most relevant in the prediction of the distribution of various invasive arthropods [43][44][45] and were used with good results in the case of predaceous true bugs 46 .Ecological niche modelling was performed with the Maxent software (version 3.4.3,November 2020) 47 .Modelling was conducted with 10 replications (replicated run type: bootstrap), and random test percentages were set to 25.All models were performed with 10000 maximum background points, regularisation multiplier 1, 1000 maximum iteration, 10− 5 convergence threshold and logistic output format following the suggestions of the above-cited articles.The accuracy and performance of the models were evaluated by using threshold-independent receiver operation characteristic (ROC) 48 analysis where the area under the curve (AUC) takes up values between 0 and 1, and models with AUC value higher than 0.75 are accepted as robust models 49 .
Distribution data and results of ENM were interpreted and maps were generated with QGIS 3.34.7 "Prizren" geographic information system using WorldClim altitude raster layer 39 and Koeppen-Geiger 1-km climatic classification maps raster layers 50 to project data.

Fig. 2 .
Fig. 2. Summary of citizen science observations on abundance, behaviour and habitat.